FRET conformational analysis of calmodulin binding to nitric oxide synthase peptides and enzymes.
Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes.
Spratt
Donald E
DE
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Taiakina
Valentina
V
Palmer
Michael
M
Guillemette
J Guy
JG
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D031541:Fluorescence Resonance Energy Transfer; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D007527:Isoenzymes; D010455:Peptides; D011994:Recombinant Proteins; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D002118:Calcium